Rhodamine derivatives for photodynamic therapy of cancer and in vitro purging of the leukemias

ABSTRACT

The present invention relates to novel photoactivable rhodamine derivatives for enhancing high quantum-yield production and singlet oxygen generation upon irradiation with light while maintaining desirable differential retention of rhodamine between normal and cancer cells. Representative derivatives are 
     2-(4,5-dibromo-6-amino-3-imino-3H-xanthen-9-yl)-benzoic acid methyl ester hydrochloride; 2-(4, 5-dibromo-6-amino-3-imino-3H-xanthen-9-yl)-benzoic acid ethyl ester hydrochloride; 2-(4,5-dibromo-6-amino-3-imino-3H-xanthen-9-yl)-benzoic acid octyl ester hydrochloride; 2-(4,5-dibromo-6-amino-3-imino-3H-xanthen-9-yl)-benzoic acid n-butyl ester hydrochloride; 2-(6-ethyl amino-3-ethyl imino-3H-xanthen-9-yl)-benzoic acid n-butyl ester hydrochloride; and photoactivable derivatives thereof. These derivatives are utilized in an amount to achieve appropriate intracellular levels of the derivative when irradiation of a suitable wavelength and intensity is applied to photoactivate the derivative and thereby induce cell killing.

This application is a divisional of application Ser. No. 08/300,179,filed Sep. 2, 1994 now U.S. Pat. No. 5,556,992.

BACKGROUND OF THE INVENTION

(a) Field of the Invention

The invention relates to a photodynamic treatment for the selectivedestruction of malignant leukemic cells without affecting the normalcells and without causing systemic toxicity for the patient.

(b) Description of Prior Art

Cancers are uncontrolled cell proliferations that result from theaccumulation of genetic changes in cells endowed with proliferativepotential. After a variable latency period during which they areclinically silent, the malignant cells progress to aggressive invasiveand metastatic stages with tumor formation, bleeding, susceptibility toinfections, and widespread dissimination throughout the body.

Despite important advances in treatment, cancers still acount for 28% ofdeath in Western countries. Treatment of cancer has relied mainly onsurgery, chemotherapy, radiotherapy and more recently immunotherapy.Significant improvement in outcome has occured with the use of combinedmodalities, for a small number of cancers. However, for the mostfrequent types of cancers (lung, breast, colo-rectal and the leukemias)complete remission and cure has not been achieved. Therefore, thedevelopment of new approaches for treating cancer patients is criticallyneeeded particularly for those patients whose disease has progressed toa metastatic stage and are refractory to standard chemotherapy. Toovercome resistance, autologous stem cell transplantation (AuSCT) hasbeen employed in the treatmemt of a number of advanced forms of cancer.Because high-dose chemotherapy with or without total body irradiationcan be applied prior to AuSCt, increased response rates have beenobserved when compared with standard chemotherapy. One important issuethat needs to be stressed when using AuSCT relates to the risk ofreinfusing residual tumor cells despite histologic remission. A varietyof techniques have been developed that can deplete up to 10⁵ of tumorcells from the marrow. These techniques, including immunologic andpharmacologic purging, are not entirely satisfactory. One majorconsideration when purging bone marrows is to preserve the normalhemopoietic stem cell compartment so that normal hemopoiesis can rapidlybecome reestablished upon grafting. The potential of photodynamictherapy, in association with photosensitizing molecules capable ofdestroying malignant cells while sparing normal hemopoietic stem cells,to purge bone marrow in preparation for AuSCT has largely beenunexplored. This issue has been investigated in depth for one type ofneoplasm, chronic myeloid leukemia (CML), since AuSCT has the potentialto cure the disease and highly sensitive molecular biologic techniquesare currently available to determine the efficacy of the purgingprocedure. Application of photodynamic therapy for treating other formsof leukemias, lymphomas and metastatic solid tumors is a distinctpossibility in view of the functional properties of the dyes moleculeswhich were synthesized in accordance with the present invention andwhose description appears below.

Chronic myeloid leukemia (CML) comprises 15% of the leukemias. It is aclonal pluripotent hemopoietic stem cell disorder characterized byderegulated proliferation of bone marrow progenitors and circulatingterminally differentiated myeloid cells. If left untreated this diseaseis invariably fatal.

Genetic analysis on CML cells has identified a highly characteristicabnormality consisting of a balanced translocation involving chromosome9 and 22 where part of the c-abl protooncogene on hromosome 9 isjuxtaposed to the 5' end of the bcr-1 gene on chromosome 22, leading tothe formation of a fusion gene, a chimeric transcript and a P210 bcr/ablprotein with tyrosine kinase activity. CML cells harboring thetranslocation are known as Ph-1+ cells, whereas non-clonal, presumablynormal but suppressed marrow cells are known as Ph-1 negative(Ph-1-)cells.

Treatment of CML patient aims at eradicating Ph-1+ cells andreestablishing non-clonal Ph-1- hemopoiesis. Conventionalmyelosuppressive therapy with hydroxyurea, busulfan, and more recentlywith interferon-alpha (IFN-alpha) and hemoharringtonine (HTT) havefailed to provide prolonged or complete clinical and cytogeneticremissions (reviewed in Goldman J. M. (1994) Blood Reviews, 8:21-29). Todate, only allogenic bone marrow transplantation (ABMT) in youngpatients (<55 years) with human leukocyte antigen-compatible(HLA-compatible) siblings donor marrow has been shown to be curative inover 50% of good risks patients. However, only a minority of patients(20%) is eligible for allogenic bone marrow transplantation because ofthe lack of suitably matched. donors or because patients are deemed tooold to withstand the procedure. Therefore, alternative strategies totreat CML had to be developed. One promising line of research that hasproduced exciting results consists of restoring Ph- hemopoiesis bygrafting patient's own marrow or peripheral blood stem cells that wereharvested in chronic phase prior to intensive chemotherapy and totalbody irradiation. This procedure, known as autologous stem celltransplantation (AuSCT) involves, no or some ex vivo marrowmanipulations to purge residual malignant Ph+ leukemic cells. To achieveeradication of the Ph-1+ clone several approaches have been proposedincluding:

1) in vitro exposure of the graft to 4-perhydroxycyclophosphamide (4-HC)or to a more stable derivative Mafosfamide™ (Asta-Z 7557);

2) in vitro selection by growth in long-term culture;

3) positive selection of CD34+DR- non-clonal stem cells; and

4) in vivo therapy with combinations of antileukemic agents or withinterferon-alpha followed by transplant.

However, the clinical relevance of these methods remains to beestablished.

There are many reports on the use of photodynamic therapy in thetreatment of malignancies (Daniell M. D., Hill J. S. (1991) Aust. N. Z.J. Surg., 61: 340-348). The method has been applied for cancers ofvarious origins and more recently for the eradication of viruses andpathogens (Raab O. (1900) Infusoria Z. Biol., 39: 524).

The initial experiments on the use photodynamic therapy for cancertreatment using various naturally occuring or synthetically producedphotoactivable substances were published early this century (JesionekA., Tappeiner V. H. (1903) Muench Med Wochneshr, 47: 2042; Hausman W.(1911) Biochem. Z., 30: 276). In the 40's and 60's, a variety of tumortypes were subjected to photodynamic therapy both in vitro and in vivo(Kessel, David (1990) Photodynamic Therapy of neoplastic disease, Vol.I, II, CRC Press. David Kessel, Ed. ISBN 0-8493-5816-7 (v. 1), ISBN0-8493-5817-5 (v. 2)). Dougherty et al. and others, in the 70's and80's, systematically explored the potential of oncologic application ofphotodynamic therapy (Dougherty T. J. (1974) J. Natl Cancer Inst., 51:1333-1336; Dougherty T. J. et al. (1975) J. Natl Cancer Inst., 55:115-121; Dougherty T. J. et al. (1978) Cancer Res., 38: 2628-2635;Dougherty T. J. (1984) Urol. Suppl., 23: 61; Dougherty T. J. (1987)Photochem. Photobiol., 45: 874-889).

Treatment of Leukemia with Photodynamic Therapy

There is currently a lack of antineoplastic agents which allow selectivedestruction of expanded leukemic cells while leaving intact the normalbut suppressed residual cellular population. Preferential uptake ofphotosensitive dye and cytotoxicity of photodynamic therapy againstleukemia cells have been previously demonstrated (Jamieson C. H. et al.(1990) Leuk. Res., 14: 209-219).

It would be highly desirable to be provided with new photosensitizerswhich possess the following characteristics:

i) preferential localization and uptake by the malignant cells;

ii) upon application of appropriate light intensities, killing thosecells which have accumulated and retained the photosensiting agents;

iii) sparing of the normal hemopoietic stem cell compartment from thedestructive effects of activated photosensitizers; and

iv) potential utilization of photosensitizers for bone marrow purging ofharvested marrow in preparation for autologous bone marrowtransplantation.

The Rhodamine Dyes

Rhodamine 123 (2-(6-amino-3-imino-3H-xanthen-9-yl) benzoic acid methylester hydrochloride), a lipophilic cationic dye of the pyrylium classwhich can disrupt cellular homeostasis and be cytostatic or cytotoxicupon high concentration exposure and/or photodynamic therapy, althoughwith a very poor quantum yield (Darzynkiewicz Z., Carter S. (1988)Cancer Res., 48: 1295-1299). It has been used in vitro as a specificfluorescent stain for living mitochondria. It is taken up and ispreferentially retained by many tumor cell types, impairing theirproliferation and survival by altering membrane and mitochondrialfunction (Oseroff A. R. (1992) In Photodynamic therapy (Henderson B. W.,Dougherty T. J. , eds) New York: Marcel Dekker, pp. 79-91). In vivo,chemotherapy with rhodamine 123 can prolong the survival of cancerousmice, but, despite initial attempts to utilize rhodamine 123 in thetreatment of tumors, its systemic toxicity of rhodamine 123 may limitits usefulness (Bernal, S. D., et al. (1983) Science, 222: 169; Powers,S. K. et al. (1987) J. Neurosur., 67: 889).

U.S. Pat. No. 4,612,007 issued on Sep. 16, 1986 in the name of RichardL. Edelson, discloses a method for externally treating human blood, withthe objective of reducing the functioning lymphocyte population in theblood system of a human subject. The blood, withdrawn from the subject,is passed through an ultraviolet radiation field in the presence of adissolved photoactive agent capable of forming photoadducts withlymphocytic-DNA. This method presents the following disadvantages anddeficiencies, The procedure described is based on the utilization ofknown commercially available photoactive chemical agents for externallytreating patient's blood, leaving the bone marrow and potential residentleukemic clones intact in the process. According to Richard L. Edelson,the method only reduces, does not eradicate, the target cell population.Moreover, the wavelength range of UV radiation used in the processproposed by Richard L. Edelson could be damageable to the normal cells.

International Application published on Jan. 7, 1993 under Internationalpublication number WO 93/00005, discloses a method for inactivatingpathogens in a body fluid while minimizing the adverse effects caused bythe photosensitive agents. This method essentially consists of treatingthe cells in the presence of a photoactive agent under conditions thateffect the destruction of the pathogen, and of preventing the treatedcells from contacting additional extracellular protein for apredetermine period of time. This method is concerned with theeradication of infectious agents from collected blood and itscomponents, prior to storage or transfusion, and does not impede on thepresent invention.

It would be highly desirable to be provided with a new approach for theuse of rhodamine derivatives in the treatment of tumors which overcomesthese drawbacks while having no systemic toxicity for the patient.

SUMMARY OF THE INVENTION

Since autologous stem cell transplantation (AuSCT) offers a potentiallycurative strategy if normal hematopoietic cells could be separated fromneoplastic stem cells either in vitro or in vivo, the possibility wasinvestigated, of using photosensitizing dyes with high quantumefficiencies of phototoxic activity in combination with photodynamictherapy (PDT) to achieve selective eradication of the Ph-1+ leukemiccells. In sharp contrast with isolated reports describing PDT-basedpurging of marrow using merocyanine-sensitized photoinactivation, themolecules of the present invention were designed to take advantage ofthe known exclusion of rhodamine-123 and its derivatives (personalobservations) from normal hemopoietic stem cells which stain poorly withrhodamine 123 and yet maintain extensive self-renewal in vitro.Moreover, because PDT-based bone marrow purging does not preclude theuse of other means of effecting positive and/or negative selection ofmarrows it could be used in conjunction with other therapeutic regimens.

One aim of the present invention is to produce new photosensitizersendowed with the following characteristics:

i) preferential localization and uptake by the malignant cells;

ii) upon application of appropriate light intensities, killing thosecells which have accumulated and retained the photosensitizing agents;

iii) sparing of the normal hemopoietic stem cell compartment from thedestructive effects of activated photosensitizers; and

iv) potential utilization of photosensitizers for bone marrow purging ofharvested marrow in preparation for autologous bone marrowtransplantation.

Accordingly, the method of he present invention using photodynamictherapy was structured so as to eradicate the malignant clonogenic cellsfrom CML bone marrow.

Another aim of the present invention is to provide, using rhodaminederivatives, a new method for the treatment of tumors which overcomesthe systemic toxicity problems, inasmuch as photodynamic therapy is usedin vitro for the purging of cancerous clones from the bone marrow ofchronic myelogenous leukemia (CML) patients.

In accordance with the present invention, the phototoxicity of rhodamineB n-butylester (2-(6-ethyl amino-3-ethyl imino-3H-xanthen-9-yl) benzoicacid n-butyl ester hydrochloride), 4,5-dibromomine 110 n-butyl ester(2-(4,5-dibromo-6-amino-3-imino 3H-xanthen-9-yl) benzoic acid n-butylester hydrochloride) and 4,5-dibromorhodamine 110 n-butyl ester(2-(4,5-dibromo-6-amino-3-imino-3H-xanthen-9-yl) benzoic acid methylester hydrochloride) and other esters (ethyl, octyl) have been assessed.

In accordance with the present invention, there is providedphotoactivable rhodamine derivatives for enhancing high quantum-yieldproduction and singlet oxygen generation upon irradiation whilemaintaining desirable differential retention of rhodamine between normaland cancer cells, said derivatives are selected from the groupconsisting of 4,5-dibromohodamine 123(2-(4,5-dibromo-6-amino-3-imino-3H-xanthen- -yl)-benzoic acid methylester hydrochloride); 4, 5-dibromorhodamine 123(2-(4,5-dibromo-6-amino-3-imino-3H-xanthen-9-yl)-benzoic acid ethylester hydrochloride); 4, 5-dibromorhodamine 123(2-(4,5-dibromo-6-amino-3-imino-3H-xanthen-9-yl)-benzoic acid octhylester hydrochloride); 4,5-dibromorodamine 110 n-butyl ester(2-(4,5-dibromo-6-amino-3-imino-3H-xanthen-9-yl)-benzoic acid n-butylester hydrochloride); Rhodamine B n-butyl ester (2-(6-ethylamino-3-ethyl imino-3H-xanthen-9-yl)-benzoic acid n-butyl esterhydrochloride); and photoactivable derivatives thereof; wherebyphotoactivation of said derivatives induces cell killing whileunactivated derivatives are substantially non-toxic to cells.

In accordance with the present invention, the complete growth inhibitionof tumor cell lines is achieved in vitro after photodynamic therapyeffected with the above-mentioned photosensitizers. This effectcontrasts with the lack of inhibitory effect upon exposure to eitherlight alone or to a saturating concentration of the photosensitizers.

Due to the specific retention of the rhodamine 123 class of dyes by theabnormal malignant cells and the concomitant lack of their accumulationby the normal hematopoietic stem cells, these results provide evidencefor the potential use of these three new dyes for in vivo or in vitrophotodynamic therapy.

BRIEF DESCRIPTION OF THE DRAWING

FIG. 1 is a graph of the photo toxicity of 4,5-dibromorhodamine 123 usedin accordance with the method of the present invention and expressed in% viability;

FIG. 2 shows three graphs 2A-2C of the photo toxicity of4,5-dibromorhodamine 110 n-butyl ester used in accordance with themethod of the present invention and expressed in % viability.

DETAILED DESCRIPTION OF THE INVENTION

Photoactive dyes are excited from the ground state to the singletexcited state following absorption of photons. Singlet excited states oforganic molecules generally have short lifetimes (10⁻¹² -10⁻⁶ sec.) asthey rapidly relax back to the ground state using non-radioactive(vibrational modes) and radioactive (fluorescence) processes.Intersystem crossing to the more stable triplet excited state is alsocompeting with relaxation to the ground state. Triplet excited statesgenerally have longer lifetimes (10-6-10 sec) which allow them todiffuse and react with other molecules in the medium.

Reactivity between molecular oxygen and a photosensitizer excited to thetriplet state, both present in malignant cells, is the operatingprinciple of most photodynamic therapies. Triplet excited states canreact with molecular oxygen via two different mechanisms. The firstmechanism (Type I) consists of the transfer of an electron from theexcited dyes to molecular oxygen, resulting in highly reactive freeradicals being present in the cellular environment.

The second mechanism (Type II) consists of the transfer of energy fromthe excited dyes to molecular oxygen, leading to the formation ofcytotoxic singlet oxygen.

Photosensitizers must therefore meet two conditions in order to be aneffective phototherapeutic agent. The first condition is that they mustbe present at a far higher concentration in malignant cells than that innormal cells. A higher concentration of dyes in malignant cells resultsin a higher concentration of photogenerate cytotoxic species andtherefore in a higher death rate. The second condition is thatirradiation of the phototherapeutic agent, in the presence ofintracellular concentrations of molecular oxygen, must lead to theformation of the cytotoxic species with high efficiency.

Rhodamine 123 is known to be taken up and preferentially retained bymany tumor cells and consequently its use as a phototherapeutic agenthas been proposed. However, the singlet excited state of Rhodamine 123does not undergo intersystem crossing to the triplet excited stateefficiently. Because of this, Rhodamine 123 is a weak phototoxinMorliere, P et al. (1990) Photochemistry and Photobiology, 52(4):703-710).

To overcome the limitations of the prior art methods, the chemicalstructure of rhodamine 123 can be modified in such a way as to enhanceintersystem crossing to the triplet excited state. Theoretically, thiscould be achieved by substituting heavy atoms, such as Br or otherhalides, for hydrogen atoms in the molecular structure of rhodamine 123.Therefore, dibromorhodamine 123 has been prepared and tested.

The amphiphatic structure and hydrophilicity of the dyes could modulatethe cytoplasmic and mitochondrial membranes and affect the phototoxicityof the dye. For example, hydrophobicity was shown to be the mostimportant factor influencing the in vitro uptake of porphyrins (Chi-WeiLin 1990) In Photodynamic therapy of neoplastic disease, Vol II, CRCPress, pp 79-101). Therefore, different esters of rhodamine 123 andrhodamine B were prepared and tested. More specifically dibromorhodaminen-butyl ester (DBBE) and rhodamine B n-butyl-ester (RBBE).

Different heavy atom substitutions of the hydrogen atoms (halogenicsubstitution) of the rhodamine backbone, for example, dibromo and diiododerivatives of rhodamine B and Rh 110, are being prepared and tested.

Dimers/oligomers, hetero dimers/oligomers of such compounds also will beprepared and tested.

Substitution of the oxygen heteroatom of the rhodamine backbone-by aheavier atom to reduce S₀ /S₁ splitting, theoritically should increasespin orbit coupling and promote intersystem crossing from the S₁ to theT₁ state, producing higher triplets yields than the original dye. Thisshould increase proportionally the production of singlet oxygen.Therefore, S (Sulfur), Se (Selenium) and Te (Tellurium) substitutionsfor the oxygen atom (O) of the rhodamine backbone is explored. Moreover, other strategies for increasing high quantum yields of Type I(free radicals) or Type II (superoxyde anion or singlet oxygen) productsand tumor selective accumulation of the dye are tested.

In accordance with the present invention, there is provided the use ofsuch above-mentioned dyes in conjugation with tumor specific antibodies,or poisonous substances, or liposomal or lipoproteins, or fluorochromeadducts.

In addition, the photosensitizes to be described have the potential toact synergistically in conjunction with other photoactive substance .

Moreover, the negative selection procedure provided by the use ofphotodynamic treatment does not preclude the use of other means forenriching hematopoietic stem cells such as positive selection withanti-CD34 monoclonal antibodies.

Other Clinical Applications

In adition to using photosensitizers in the context of in vitro bonemarrow purging for the leukemias and metastatic cancers, the moleculescan also be used in vivo for tumor sites directly accessible to exposureto a light source and to appropriate local concentrations of the drugsto be described. The molecules of the invention can also be utilized inthe photodynamic therapy of a patient suffering from disseminatedmultiple myelomas or lymphomas. The metastatic cancers for which thetherapy of this invention is appropriate include metastasis of breast,lung, prostatic, pancreatic and colonic carcinomas, disseminatedmelanomas and sarcomas. The photoactivable derivatives of the presentinvention can be administered by instillation, injection, bloodstreamdiffusion at the tumor sites directly accessible to light emission ortumor sites accessible to laser beams using rigid or flexibleendoscopes.

Chemical Synthesis

All flash chromatography was done according to the method of Still etal. (Still W. C. et al. (1978) J. Org. Chem., 43: 2923). Thin-layerchromatography was conducted on silica Gel 60™ (HF-245, E. Merck) at athickness of 0.20 mm. Nuclear magnetic resonnance spectra were obtainedwith a Varian VXR 300™ (300 MHz) instrument. Spectral data are reportedin the following order: chemical shift (ppm), multiplicity, couplingconstants, number of proton, assignment. Low resolution mass spectrausing fast atom bombardment (FAB), were obtained on a Kratos MS-50 TA™spectrometer. Ultraviolet spectra were obtained on a Varian DMS100™spectrophotometer and data are presented as λ/max.

1. Preparation of rhodamine B n-butylester ##STR1##

Rhodamine B hydrochloride (150 mg, 0.31 mmol) was dissolved in 1-butanol(5 ml). The reaction mixture was saturated with HCl (gas) and thenstirred at 100° C. for 15 hr. 1-Butanol was evaporated under reducedpressure. The crude oily residue was purified by flash chromatographyusing CH₂ Cl₂ (200 ml) and then CH₂ Cl₂ /CH₃ OH (85:15) as eluantyielding 142 mg (0.27 mmol, 87% yield) of a dark red solid.

¹ H NMR (Varian 300 MHz, Acetone, TMS) d 8.31 (dd, J=1.4 and 7.8 Hz,1H); 7.86-7.94 (M, 2HO); 7.54 (dd, J=1.5 and 7.4 Hz, 1H); 7.14-7.23 (M,4H); 7.02 (d, J=2.2 Hz, 2H); 3.97 (t, J=6.3 Hz, 2H); 3.79 (q, J=7.1 Hz,8H); 1.32 (t, J=7.1 Hz, 12H); 1.2-1.4 (M, 2H); 1.01 (h, J=7.5 Hz, 2H);0.75 (h, J=7.3 Hz, 3H). UV (methanol)/_(max) : 545 nm

2. Preparation of dibromorhodamine n-butylester ##STR2## 2.1 Preparationof rohodamine n-butylester

Rhodamine 110 (14 mg, 0.038 mmol) was dissolved in 1-butanol (5 ml). Thereaction mixture was saturated with HCl (gas) and then stirred at 100°C. for 15 hr. The 1-Butanol was evaporated under reduced pressure. Thecrude oily residue was purified by flash chromatography using CH₂ Cl₂/CH₃ OH (85:15) as eluant yielding 14 mg (0.033 mmol, 87% yield) of ared solid.

2.2 Preparation of dibromorhodamine n-butylester

Rhodamine n-butylester (14 mg, 0.033 mmol) was dissolved in absoluteethanol (3 ml), then bromine (0.0036 ml, 0.070 mmol) was added. Themixture was stirred at room temperature for 1 hr. The solvent wasevaporated and the crude reaction residue was purified by flashchromatography using CH₂ Cl₂ /CH₃ OH (85:15) as eluant yielding 15.9 mg(0.027 Mol, 83% yield) of a red solid.

¹ H NMR (Varian 300 MHz, CD₃ OD) d 8.31 (dd, J=1.7 and 7.5 Hz, 1H); 7.84(M, 2H); 7.46 (dd, J=1.8 and 6.9 Hz, 1H); 7.12 (d, J=9.2 Hz, 2H); 7.03(d, J=9.2 Hz, 2H); 3.95 (t, J=6.2 Hz, 2H); 1.22 (M, 2H); 0.93 (M, 2H);0.75 (t, J=7.3 Hz, 3H). MS (LR,FAB) m/z; Calculated for C₂₄, H₂₁ N₂ O₃Br₂ ; 543 Observed: 543

3.Preparation of dibromorhodamine 123 ##STR3##

To a solution of rhodamine 123 (25 mg, 0.066 mmol) in dry ethanol (1ml), was added bromine (0.01 ml, 0.19 mmol) and the resulting mixturewas stirred at room temperature for 0.5 hr. Evaporation of solvent invacuum provided the crude compound which was purified by flashchromatography using CH₂ Cl₂ /CH₃ OH (85:15) as eluant yielding. 27.0 mg(0.050 Mol, 77% yield) of a red solid.

¹ H NMR (Varian 300 MHz, CD₃ OD) d 8.34 (dd, J=1.7 and 7.5 Hz, 1H); 7.85(M, 2H); 7.46 (dd, J=1.7 and 7.2 Hz, 1H); 7.10 (d, J=9.2 Hz, 2H); 7.01(d, J=9.2 Hz, 2H); 3.64 (s, 3H). 8.3 (d, 1H, 9.1 Hz, aromatic), 7.9 (m,2H,aromatic), 7.45 (d, 1H, 9.1 Hz, aromatic), 7.0, 7.2 (AB system, 4H,aromatic), 3.64 (s, 3H, OCH₃). MS (LR, FAB) m/z: Calculated for C₂₄, H₂₁N₂ O₃ Br₂ ; 501 Observed: 501 UV (methanol)/_(max) : 510 nm

Physical and Photochemical Properties

After synthesis, the purity of the preparation of the dyes was assessedby NMR analysis and was shown to be over 99.9%. Absorption and emissionspectra were determined for each dye.

Evaluation of Cell Viability

The K562 chronic myelogeneous leukemia cell line. (Lozzio, B. B. andLozzio, C. B- (1979) Cancer Res., 3(6); 363-370) was obtained from theAmerican Type Culture Collection (ATCC, 12301 Parklawn Drive, Rockville,Md. 20852 USA) under the accession number F-10601 R243-CCL. Cultureswere maintained at 37° C. in a humidified incubator with an atmosphereof 95% air and 5% CO₂. The culture media (IMDM (Iscove Modified DulbecoMedia) supplemented with 10% fetal bovine serum) were changed bi-weeklyand the cells resuspended at a concentration of 100,000/ml. Cells wereshown to be mycoplasma negative by routinely testing at 4 weeksinterval.

Before each experiment, cell viability was assessed and 2×10⁶ viablecells were distributed in each 15 ml test tube. Cells were thenincubated for 1 hour at 37° C. , spun down and the cell pelletresuspended in the culture media in the presence or absence of dye. Thecells were then incubated for the appropriate time at 37° C., generally40 minutes, then washed twice in PBS (Phosphate Buffer Saline) andresuspended in the culture media. Photodynamic therapy was then appliedto the cell culture, immediately or after an incubation period at 37° C.The cell cultures were kept at 4° C. during the application ofphotodynamic activation.

Phototoxicity of 4,5-Dibromorhodamine 123

To assess the photochemotherapeutic potential and the in vitrophototoxicity of 4,5-dibromorhodamine 123 (DBR), the leukemic K-562 cellline assay (as described above) was applieded. Exposure to 514.5 nmradiation from an argon ion laser at 10 J/cm² induced photo toxicity inK-562 cells treated with DBR at a final concentration of 10 μg/ml. DBRwas shown to be markedly more phototoxic than rhodamine 123; theincreased activity is beleived to be a consequence of increasedintersystem crossing of DBR to the triplet manifold via spinorbital-coupling induced by the heavy atoms. As shown in FIG. 1,dibromorhodamine is extremely phototoxic at doses as little as 1 J/cm²and the cell viability drops under 5% in less than 24 hours afterirradiation.

Phototoxicity of 4,5-Dibromorhodamine 110 n-Butyl Ester

To ascertain the photochem therapeutic potential of 4,5-dibromorhodamine110 n- butyl ester (DBBE), in vitro phototoxicity was evaluated in theK-562 cell line procedure described. The cells were incubated withincreasing concentrations of DBBE and the cell viability was measured atdifferent time points following photodynamic therapy. The results shownin FIG. 2 show that a dosage of 10 μg/ml of the dye and a brief exposureat 0.5 J/cm² completely suppress cell viablity in less than 24 hoursafter irradiation.

Photo Toxicity of Rhodamine B n-Butyl Ester

The photo toxicity in vitro of rhodamine B n-butyl ester (RBBE) wasevaluated in the K-56 2 cell line procuedure, in order to as access itsphotochemotherapeutic potential. Comparison was made to the inducedphototoxicity of rhodamine 123 (123RH) and of rhodamine B butyl ester.Cell viability was evaluated 2 and 20 hours after photodynamic therapy.The results shown in FIG. 3 demonstrate that a dosage of 10 μg/ml of thedye and a photo exposure of 5 J/cm² significantly suppress cell viablityof K562 cells in less than 20 hours after irradiation. Rhodamine 123 hasno effect on cell viability, even at exposures of 10 J/cm².

Phototoxcitity Against Bone Marrow Cultures

It is observed that the photo treatment alone, at energy levels up to 10J/cm2, or the pre-incubation of the cells at saturating concentrationsof the dyes did not affect neither the estabishment of the long termculture nor the formation in semi solid assays of cellular coloniesissued from the multiplication and differentiation of committedprogenitors present in the bone marrow (colony formingunits-erythrocytes (CFU-E), blast forming units-erythrocytes (BFU-E),colony forming units-granulocytes, macrophages, (CFU-G-M)). However, asreported for rhodamine 123, the LTC (Long Term Culture) establisment ismore sensitive to the dyes but the number of viable commited precursorand stem cells remains unaffected. Photodynamic therapy withrhodamine123, rhodamine B n-butyl ester and 4,5-dibromorhodamine n-butylester minimally impaired the establishment of normal mouse long termculture of bone marrow and the formation of hematopoietic colonies insemi-solid assays. This is in agreement with results obtained previouslyin other laboratoties using rhodamine 123.

Conventional approaches for the treatment of cancer such as radiotherapyand intensive chemotherapy are limited by their intrinsic toxicity andmyelosuppressive effects. The introduction of allogeneic and autologousbone marrow transplantation have allowed the administration of marrowablative chemotherapy and radiotherapy to patients whose malignanciescannot be cured with less aggressive measures. However, allogeneic bonemarrow transplantation is not widely accessible to patients because ofthe lack of suitable donnors and the onset of graft-versus-host diseasein recipients. To overcome these limitations and to expand the number ofpatients and age limit for intensive curative therapy, the potentialbenefit of in vitro bone marrow purging and autologous bone marrowtransplantation has become widely acknowledged.

In an effort to develop new anti-neoplastic drugs that would allowselective destruction of leukemic malignant cells, new dye moleculeshave been prepared and tested as possible new photosensitizers, usefulfor the photodynamic therapy of leukemias and metastatic cancers. Threenew photosensitizers of the pyrylium family were prepared and there isprovided evidence for their potential use in the photodynamic treatmentof cancers and the leukemias.

The present invention will be more readily understood by referring tothe following examples which are given to illustrate the inventionrather than to limit its scope.

EXAMPLE I Method of treatment of leukemias

1. Diagnostic procedures

Diagnosis of chronic myelogenous leukemia (CML) will be establishedusing one or more of the following procedures on blood or bone marrowcells:

a) conventional cytogenetics studies with identification of Ph 1+metaphases harbouring the t(9:22);

b) fluorescent in situ hybridization for the detection of the bcr/ablrearrangement; and

c) Southern blot analysis for the detection of a rearranged ber fragmentor PCR-RT for the detection of chimeric ber/abl messenger RNA.

2. Bone marrow harvesting

After diagnosis, bone marrow (BM) or peripheral blood (PB) derivedhemopoietic stem cells will be harvested using previously describedprocedures for the autologous marrow transplantation in cancer therapy(reviewed by Herzig GP, (1981) Prog. Hematol., 12:1). Hemopoietic stemcells collected for autograft will be immediately treated ex vivo asdescribed below.

3. In vitro purging of leukemia

Ex vivo treatment will consist of short-term incubation or BM of PB stemcells with one or several of the selected photoactive compounds.Duration of incubation, cell concentration and drug molarity will bedetermined for each patient using an aliquot of the harvested cellpopulation. Excess of dyes will be removed by cell washes with steriledye free medium supplemented with 2% autologous serum. Cells will nextbe exposed to radiant energy of sufficient intensities to effectphotodynamic purging of leukemia cells. Efficacy of the photodynamicpurging procedure will be verified on an aliquot of the treated cellpopulation, before cryopreservation and/or re-infusion to the patient isperformed. Until re-infusion to the patient, the cells will becryopreserved in 10% dimethyl sulfoxyde (DMSO)--90% autologous serummedium,. at -196° C. in the vapour phase of liquid nitrogen.

4. Systemic treatment of patients

Following stem cell harvest, patient will be either treated withconventional regimens until autografting is clinically indicated orimmediately submitted to dose-intensive chemotherapy and total bodyirradiation where indicated.

5. Autologous stem cell transplantation

Following appropriate treatment of the patient by high-dose chemotherapyand irradiation and at the appropriate clinical moment, cryopreservedmarrow or peripheral blood stem cells will be rapidly thawed and dilutedin medium containing 25 UI DNase ml⁻¹ to minimize clumping. A minimum of2×10⁷ /kg nucleated cells with 85% to 95% viability as measured byTrypan™ blue exclusion will be returned to the patient.

EXAMPLE II Method of treatment of malignancies

1. Diagnostic procedures

Diagnosis of malignancies will be established using conventionalhistopathological examination of the primary tumor. Detection of marrowinvolvement by neoplastic cells will be achieved by direct histologicalexamination and ancillary procedures where indicated (i.e.immuno-peroxydose, immunohistochemical, tumor markers and hybridizationstudies).

2. Bone marrow harvesting

After diagnosis, bone marrow (BM) or peripheral blood (PB) derivedhemopoietic stem cells will be harvested using previously describedprocedures for the autologous marrow transplantation in cancer therapy(reviewed by Herzig GP, (1981) Prog. Hematol., 12:1). Hemopoietic stemcells collected for autograft will be treated immediately ex vivo asdescribed below.

3. In vitro purging of leukemia

Ex vivo treatment will consist of short-term incubation of BM of PB stemcells with one or several of the selected photoactive compounds.Duration of incubation, cell concentration and drug molarity will bedetermined for each patient using an aliquot of the harvested cellpopulation. Excess of dyes will be removed by cell washes in steriledye, free medium supplemented with 2% autologous serum. Cells will nextbe exposed to radiant energy of sufficient intensities to effectphotodynamic purging of leukemia cells. Whenever a sensitive molecularmarker is available, an aliquot of the treated cell population will betested for the detection of residual neoplastic cells beforecryopreservation and/or re-infusion to the patient is attempted. Thecells will be cryopreserved in 10% dimethyl sulfoxyde (DMSO)--90%autologous serum medium, at 196° C. in the vapour phase of liquidnitrogen.

4. Systemic treatment of patients

Following stem cell harvest, patient will be either treated withconventional regimens until autografting is clinically indicated orimmediately submitted to dose-intensive chemotherapy an total bodyirradiation where indicated.

5. Autologous stem cell transplantation

Following high-dose chemotherapy and irradiation cryopreserved marrow orperipheral blood stem cells will be rapidly thawed and diluted in mediumcontaining 25 UI DNase Ml⁻¹ to minimize clumping. A minimum of 2×10⁷ /kgnucleated cells with 85% to 95% viability as measured by Trypan™ blueexclusion will be returned to the patient.

While the invention has-been described in connection with specificembodiments thereof, it will be understood that it is capable of furthermodifications and this application is intended to cover any variations,uses, or adaptations of the invention following, in general, theprinciples of the invention and including such departures from thepresent disclosure as come within known or customary practice within theart to which the invention pertains and as may be applied to theessential features hereinbefore set forth, and as follows in the scopeof the appended claims.

We claim:
 1. A photodynamic pursuing method of treating a cancer patientto destroy human cancer cells which comprises administration of aphotoactivable rhodamine derivatives selected from the group consistingof 2-(4,5-dibromo-6-amino-3-imino-3H-xanthen-9-yl)-benzoic acid methylester hydrochloride;2-(4,5-dibromo-6-amino-3-imino-3H-xanthen-9-yl)-benzoic acid ethyl esterhydrochloride; 2-(4,5-dibromo-6-amino-3-imino-3H-xanthen-9-yl)-benzoicacid n-butyl ester hydrochloride; 2 -(6-ethyl amino-3-ethylimino-3H-xanthen-9-yl)-benzoic acid n-butyl ester hydrochloride; in anamount to achieve appropriate intracellular levels of said derivativeand application of irradiation of a suitable wavelength and intensity tophotoactivate said derivative to induce cell killing.
 2. The methodaccording to claim 1 for a photodynamic therapy of a patient sufferingfrom leukemias, disseminated multiple myelomas or lymphomas, whichcomprises the steps of:a) harvesting said patient's human bone marrow;b) purging of the bone marrow of step a) by photodynamic therapy using atherapeutic amount of said photoactivable derivative under irradiationof a suitable wavelength; and c) performing autologous stem celltransplantation using the purged bone marrow of step b).
 3. The methodof claim 2, wherein said purging of step b) further comprises intensivechemotherapy and total body irradiation (TBI) procedures.
 4. A methodaccording to claim 1 for in vitro purging of the human bone marrowcontaining metastasis of solid tumors, selected from the groupconsisting of metastasis of breast, lung, prostatic, pancreatic andcolonic carcinomas, disseminated melanomas and sarcomas, whereinsurgical excision or debulking can be achieved, which comprises thesteps of:a) harvesting said patient's human bone marrow; b) purging ofthe bone marrow of step a) by photodynamic therapy using a therapeuticamount of said photoactivable derivative under irradiation of a suitablewavelength; and c) performing autologous stem cell transplantation usingthe purged bone marrow of step b).
 5. The method of claim 4, whereinsaid purging of step b) further comprises intensive chemotherapy andtotal body irradiation (TBI) procedures.
 6. The method of claim 1,wherein said photoactivable derivative is administered by instillation,injection, bloodstream diffusion at the tumor sites directly accessibleto light emission or tumor sites accessible to laser beams using rigidor flexible endoscopes.
 7. The method of claim 6, wherein saidlaser-accessible tumor site is selected from the group consisting ofurinary bladder, oral cavity, esophagus, stomach, lower digestive tract,upper and lower respiratory tract.
 8. A photodynamic purging method forthe treatment of leukemias in a patient, which comprises the steps of:a)purging of cancerous clones from the bone marrow of said patient; b)subjecting said purged clones of step a) to a photodynamic treatmentusing a therapeutical amount of the photoactivable derivatives selectedfrom the group consisting of 2-(4,5-dibromo-6-amino-3-imino-3H-xanthen-9-yl)-benzoic acid methyl ester hydrochloride;2-(4,5-dibromo-6-amino-3-imino-3H-xanthen-9-yl)-benzoic acid ethyl esterhydrochloride; 2-(4,5-dibromo-6-amino-3-imino-3H-xanthen-9-yl)-benzoicacid octyl ester hydrochloride; 2-(4,5 -dibromo-6-amino-3 -imino-3H-xanthen-9-yl)-benzoic acid n-butyl ester hydrochloride; 2-(6-ethylamino-3-ethyl imino-3H-xanthen-9-yl)-benzoic acid n-butyl esterhydrochloride; and photoactivable derivatives thereof; under irradiationof a suitable wavelength for the selective destruction of leukemic cellswithout affecting the normal cells of the patient; c) administering saidclones of step b) to the patient;thereby causing no systemic toxicityfor the patient.
 9. The method according to claim 1 which comprises theadministration of the rhodamine derivative which is2-(4,5-dibromo-6-amino-3-imino-3H-xanthen-9-yl)-benzoic acid methylester hydrochloride.
 10. The method according to claim 1 which comprisesthe administration of the rhodamine derivative which is2-(4,5-dibromo-6-amino-3-imino-3H-xanthen-9-yl)-benzoic acid ethyl esterhydrochloride.
 11. The method according to claim 1 which comprises theadministration of the rhodamine derivative which is2-(4,5-dibromo-6-amino-3-imino-3H-xanthen-9-yl)-benzoic acid octyl esterhydrochloride.
 12. The method according to claim 1 which comprises theadministration of the rhodamine derivative which is2-(4,5-dibromo-6-amino-3-imino-3H-xanthen-9-yl)-benzoic acid n-butylester hydrochloride.
 13. The method according to claim 1 which comprisesthe administration of the rhodamine derivative which is 2-(6-ethylamino-3-ethyl imino-3H-xanthen-9-yl)-benzoic acid n-butyl esterhydrochloride.